Capability of Cartilage Extract to In Vitro Differentiation of Rat Mesenchymal Stem Cells (MSCs) to Chondrocyte Lineage.

The importance of mesenchymal stem cells (MSCs), as adult stem cells (ASCs) able to divide into a variety of different cells is of utmost importance for stem cell researches. In this study, the ability of cartilage extract to induce differentiation of rat derived omentum tissue MSCs (rOT-MSCs) into chondrocyte cells (CCs) was investigated. After isolation of rOT-MSCs, they were co-cultured with different concentrations of hyaline cartilage extract and chondrocyte differentiation was monitored. Expression of MSCs markers was analyzed via flow cytometry. Moreover, expression of octamer- binding transcription factor-4 (Oct-4), Wilm's tumor suppressor gene-1 (WT-1), aggrecan (AG), collagen type-II (CT-II) and collagen type-X (CT-X) was analyzed using RT-PCR on 16, 18 and 21 days. Furthermore, immunocytochemistry and western blot were performed for CT-II production. Finally, proteoglycans (PGs) were examined using toluidine blue and alcian blue staining. The phenotypic characterization revealed the positive expression of CD90, CD44 and negative expression of CD45 in rOT-MSCs. These cells also expressed mRNA of Oct-4 and WT-1 as markers of omentum tissue. Differentiated rOT-MSCs in the presence of 20 µg/ ml cartilage extract expressed AG, CT-II, CT-X, and PGs as specific markers of CCs. These observations suggest that cartilage extract is potentially able to induce differentiation of MSCs into chondrocyte lineage and may be considered as an available source for imposing tissue healing on the damaged cartilage. More investigations are needed to prove in vivo cartilage repair via cartilage extract or its effective factors.

implantation (ACI) has been used clinically for more than 20 years. However, extensive applications are limited by intrinsic problems.
Among these, cell source shortage is a key issue that needs to be resolved by exploring other resources such as stem cells (3)(4). MSCs can be isolated from bone marrow, synovium, periosteum, skeletal muscle and adipose tissue (5)(6)(7). In vitro chondrogenesis of MSCs has been investigated with the use of growth factors and cytokines, the microenvironment, including TGF-β, BMP-6, dexamethasone and insulin (8)(9). Omentum is considered as a source of ASCs and the non-fat stromal cells in the expanded omental tissue, express markers of stem cell activity, such as stromal cell derived factor 1 (SDF1-α), chemokine receptor 4 (CXCR4), and Wilms' tumor antigen 1 (WT-1) (10). Therefore, cultured omental cells could represent a readily available source of MSCs that could be used to repair and regenerate damaged tissue. Omental stromal cells (OSCs), however, are more easily obtainable in large quantities, can be harvested from the patient's own omentum, and able to passage in culture and differentiates into target tissues (11,12). Oct4 is critically involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells (13). Based on the well-known healing property of the MSCs, cultured OSCs could qualify as potential stem cells from the adult for articular cartilage (14). If so, the omentum would be a convenient source of ASCs that could be used to repair and possibly regenerate damaged tissues (15,16). Cell treatement with tissue extract or nutrient supplements is a new strategy for differentiation, with the lowest cost (17,18). Some researchers have emphasized that local environment and resident cellular populations are the major factors determining the fate of engrafted cells (19). Also, cell extracts may prove useful for investigating the molecular mechanisms of stem cell differentiation . The cultured MSCs also express   on their surface CD73, CD90, CD44 and CD105,   while lacking the expression of CD11b, CD14, CD19, CD34, CD45 and CD79a surface markers (20,21). Hyaline cartilage extract is rich in different growth factors and molecules that are effective in their proliferation and differentiation (1). In this study, we decided to monitor differentiation of isolated rOT-MSCs to chondrocytes in the presence of hyaline cartilage extract.

Materials and methods
Isolation, culture, expansion and storage of rOT-

MSCs
Cell isolation was performed according to

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
RT-PCR analysis was performed for specific genes on days 16, 18 and 21 according to standard protocol with some modifications (25). Total RNA was isolated from cell lysates using TriPure The RT-PCR procedure was carried out in one step using 1 µg of total tissue RNA and random hexamers as primers using the RT-PCR system (12,13,26  gelatin, followed by HRP-conjugated secondary antibody (HRP-labelled goat anti-rat collagen type II IgG). Immunoreactive bands were developed by ECL (Amersham) and blots were exposed to medical X-ray film (Kodak, Rochester, NY,USA).
The specific bands were detected by using the

Statistical analyzes
The data presented were means of triplicate determinations. One-way ANOVA test was used for statistical analysis of more than one group of samples. For all other data, unpaired t-test was used. A p-value of, 0.05 was considered statistically significant.

rOT-MSCs culture and characterization
The initial adherent spindle-shaped rOT-MSCs appeared as separate colonies at the bottom of culture flasks within 19 h (Fig. 1a). Cells in 2-5 days culture became more confluent (Fig. 1b and c), and reached 75-80% of confluence within one week (Fig. 1d). Morphologically verified cells were lifted and cultured in another 25 cm 2 flasks (1:3) until confluence was achieved. Cell population appeared to be more homogeneously formed of spindleshaped cells (Fig. 1e) and following a freezethaw stage these cells reattached to the culture flasks with slow growth rate (Fig. 1f). Molecular analysis (RT-PCR) confirmed that the isolated cells express omental (Oct-4 and WT-1) markers ( Fig. 2). Moreover, expression of cell surface mesenchymal markers (CD90 and DC44) was confirmed via flow cytometry assay (Fig. 3). Cells were let to attach and adapt with new environment during first days and then flow cytometry analysis was performed after one week treatment (75-80% of confluence).
Flow cytometry analysis revealed that the rOT-MSCs were positive for CD90 (84.05%), CD44 (88.22%) and relatively negative for CD45 (1.02%) (Fig. 3). compared with rOT-MSCs as control (Fig. 4). The morphology of chondrocyte-like cells were observed as early as 18 days after culturing under cartilage extract and further matured by day 21 in the presence of 2% FBS (Fig. 4).

Characterization of chondrocyte-like cells
Differentiation of cells to the chondrocyte phenotype was confirmed with expression of aggrecan (AG), collagen type-II (CT-II) and collagen type-X (CT-X) (Fig. 2). However, CT-II,

II (CT-II)
The presence of CT-II is a prominent feature of mature chondrocyte, as cartilage is the predominant site for the synthesis of CT-II protein.
The ICC results showed that the cells expressed whereas a small amount of CT-II was present in the media of undifferentiated (control) cells (Fig. 6A).
The rate of CT-II secretion by the differentiated cells was 75 to 95 times higher than undifferentiated or control cells (Fig. 6A).

Toluidine blue and alcian blue staining for proteoglycans (PGs)
Proteoglycan production was studied for  6B). The positive rates were 60% to 70%, while undifferentiated MSCs were negative (Fig. 6B).

Discussion
Cartilage tissue damages are a rather worldwide problem of many people and are produced by either trauma or age dependent degenerative diseases (28). MSCs could be considered as an appropriate source for cell-based treatment of cartilage defects owing to their capacity in undergoing extensive self-renewal proliferation as well as the potential of giving rise to chondrocyte cell lineage (29). The omentum has long been known to have the power to heal injured organs once it has adhered to the damaged site, either naturally or deliberately by surgery (10). It has been previously shown that the omentum, especially after its activation by injury, becomes a reservoir of stromal cells that express stem cell markers and growth factors (16,30). At present,